Nonalcoholic fatty liver (hepatic steatosis) can progress by unknown mechanisms to nonalcoholic steatohepatitis (NASH) that is characterized by hepatocyte injury and death, inflammation and fibrosis. Both human NASH and animal models of NASH are associated with increased expression of the pro-oxidant enzyme cytochrome P450 2E1 (CYP2E1). We hypothesize that sustained CYP2EI expression promotes the progression from steatosis to NASH through oxidant-mediated alterations in cell death signaling pathways that sensitize hepatocytes to injury and death from NASH-related cofactors such as tumor necrosis factor-a (TNF-ct) and polyunsaturated fatty acids. To test this hypothesis we have developed novel, nontransformed hepatocyte cell lines with differential CYP2E1 expression. Preliminary studies have demonstrated that increased CYP2E1 expression sensitizes hepatocytes to death stimuli in association with alterations in mitogen-activated protein kinase signaling, transcription factor activation, and cellular glutathione content. The goal of this proposal is to determine the molecular mechanisms by which increased CYP2E1 expression promotes hepatocyte death, and contributes to the progression from steatosis to NASH. The specific aims of this proposal are to: (1) Determine the mechanism by which increased CYP2E1 expression sensitizes hepatocytes to injury and cell death from TNF-a and polyunsaturated fatty acids. (2) Identify AP-1 transcriptional activation as the ultimate downstream effector of cell death in CYP2E1-expressing hepatocytes. (3) Define cellular changes induced by CYP2E 1 expression that promote cell death from necrosis rather than apoptosis. (4) Examine the ability of lipopolysaccharide and TNF-a to trigger the progression to steatohepatitis in an in vivo NASH model. The ultimate objective of these investigations is to better understand the mechanisms leading to the progression of steatosis to steatohepatitis, in order to develop new therapies to prevent the hepatocyte injury that underlies human NASH.